A conditional suicide system in Escherichia coli based on the intracellular degradation of DNA

Abstract

The potential risks associated with the intentional or unintentional release of genetically engineered microorganisms led to the construction of biological containment systems by which bacteria are killed in a controlled suicide process. In previously published suicide systems, cell killing was caused by proteins destroying the cell membrane or cell wall. Here a conditional cell killing system based on the intracellular degradation of cellular DNA is presented. The nuclease gene used was that of the extracellular nuclease of Serratia marcescens. The nuclease gene was deleted for the leader-coding sequence, and the truncated gene was put under the control of the lambda PL promoter. Following thermoinduction of the nuclease gene cassette in Escherichia coli, cell survival dropped to 2 x 10i, and more than 80% of the radioactively labeled DNA was converted to acid-soluble material within 2.5 h in the absence of cell lysis. The majority (84%) of clones which survived thermoinduced killing turned out to be as sensitive to a second thermoinduction as the original strain. The other clones showed somewhat slower killing kinetics or slightly higher final levels of survivors. The suicide system described combines the regulated killing of cells with the destruction of intracellular DNA otherwise potentially available for horizontal gene transfer processes.

Growth and productivity impacts of periplasmic nuclease expression in an Escherichia coli Fab' fragment production strain

“…We previously used periplasmic nuclease activity for disruption‐actuated removal of host nucleic acids from E. coli homogenates at laboratory (Cooke et al, 2003) and pilot scale (Balasundaram et al, 2009). Nuclease expression in the E. coli cytosol is toxic (Ahrenholtz et al, 1994) due to hydrolysis of the host cell genome. Periplasmic nuclease localization ensures non‐toxicity during growth as the inner cell membrane protects the host genome from the nuclease enzyme.…”

Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology

“…by RecBCD helicase-nuclease and other endogenous nucleases (15,19,20). DSBs were made via I-CeuI endonuclease, which recognizes a specific 26-base pair sequence (5′-TAACTATAAC-GGTCCTAAGGTAGCGA-3′) (21).…”

Serratia marcescensand its extracellular nuclease

“…Therefore the release of genetically engineered organisms can be controlled, environmental signals triggering the expression of such a toxic cassette results in suicide. There are of course numerous such suicide systems, however the nuclease system has the added advantage of destroying any recombinant DNA plasmids as well, thereby alleviating fears concerning the release of such DNA [64].…”